Flow cytometry is a technology used to analyze the physical and chemical characteristics of cells or particles in horses. This technique involves suspending cells in a fluid stream and passing them through a laser beam, where they are individually measured for various parameters such as size, complexity, and fluorescence intensity. In equine research, flow cytometry is applied to study immune cell populations, assess cellular responses to pathogens, and evaluate hematological parameters. It is a valuable tool for veterinary diagnostics, allowing for detailed analysis of blood and tissue samples. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and findings related to flow cytometry in equine health and disease.
Hamza E, Steinbach F, Marti E.Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Cu...
Corradetti B, Lange-Consiglio A, Barucca M, Cremonesi F, Bizzaro D.Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Methods: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes...
Dougherty SS, Santangelo KS, Bertone AL.To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses. Methods: Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses. Methods: Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested...
Moran G, Buechner-Maxwell VA, Folch H, Henriquez C, Galecio JS, Perez B, Carrasco C, Barria M.Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition similar to human asthma caused by exposure of susceptible horses to poorly ventilated stable environments. The disease is characterized by neutrophilic airway inflammation, mucus hypersecretion and reversible bronchoconstriction. This inflammatory process is mediated by several factors, including antibodies, cytokines, resident cells of the airway and inflammatory cellular components that arrive in the respiratory tract. An increasing body of evidence has lent support to the concept that a dysregulatio...
Kwon S, Gewirtz AT, Hurley DJ, Robertson TP, Moore JN, Vandenplas ML.As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species...
Oldenhof H, Blässe AK, Wolkers WF, Bollwein H, Sieme H.The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1...
Foster ML, Love CC, Varner DD, Brinsko SP, Hinrichs K, Teague S, Lacaze K, Blanchard TL.Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Blan...
da Silva CM, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G....Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at ...
Breuer J, Müller U, Locher L, Spallek A, Recknagel S, Uhlig A, Schusser GF.Proinflammatoric cytokines are released extracellularly during necrosis. These lead to inflammation and destruction of surrounding tissues. The aim of this study was to compare the number of viable, apoptotic and necrotic cells in the bronchoalveolar lavage fluid (BALF) of normal horses and horses with recurrent airway obstruction (RAO) and to determine if fluorescence microscopy is a reliable method for this examination. A group of six normal horses and a group of ten horses with RAO were examined. Samples were assessed using annexin-V and propidium iodide immunofluorescence assay and examine...
Doty A, Buhi WC, Benson S, Scoggin KE, Pozor M, Macpherson M, Mutz M, Troedsson MH.Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff)...
Park J, Sutradhar BC, Hong G, Choi SH, Kim G.To compare the chondrotoxicity of bupivacaine, lidocaine, and mepivacaine in equine articular chondrocytes in vitro. Methods: Prospective, experimental study. Methods: Equine articular chondrocytes. Methods: Primary cultured equine chondrocytes were exposed to 0.5% bupivacaine, 2% lidocaine, or 2% mepivacaine for 30 or 60 minutes. After treatment, cell viability was evaluated by trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in a dose dependent manner. Apoptosis and necrosis of chondrocytes were analyzed with the double stain...
Foster ML, Varner DD, Hinrichs K, Teague S, Lacaze K, Blanchard TL, Love CC.Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and a...
Macías García B, González Fernández L, Ortega Ferrusola C, Morillo Rodríguez A, Gallardo Bolaños JM, Rodríguez Martinez H, Tapia JA....Fatty acids and plasmalogens were extracted from the phospholipids of the plasma membrane of stallion spermatozoa, to determine their relation with sperm quality after freezing and thawing. Sperm quality was rated using a quality index that combined the results of the analysis of sperm motility and velocity (CASA analysis), membrane status and mitochondrial membrane potential (flow cytometry) post thaw. Receiving operating system (ROC) curves were used to evaluate the value of specific lipid components of the sperm membrane herein studied as forecast of potential freezeability. From all parame...
Gibb Z, Morris LH, Maxwell WM, Grupen CG.The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 (H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohn's Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after co...
Robbin MG, Wagner B, Noronha LE, Antczak DF, de Mestre AM.Several distinct T lymphocyte subpopulations with immunoregulatory activity have been described in a number of mammalian species. This study performed a phenotypic analysis of cells expressing regulatory T cell (Treg) markers in the peripheral blood of a cohort of 18 horses aged 6 months to 23 years, using antibodies to both intracellular and cell surface markers, including Forkhead box P3 (FOXP3), CD4, CD8, CD25, interferon gamma (IFNγ) and interleukin 10 (IL-10). In peripheral blood, a mean of 2.2 ± 0.2% CD4+ and 0.5 ± 0.1% CD8+ lymphocytes expressed FOXP3. The mean percentage of CD4+FOXP...
Wauters J, Martens A, Pille F, Dumoulin M, Gasthuys F, Sys S, Meyer E.Synovial fluid samples from culture-confirmed infected joints (n=13), joints with pronounced non-infectious synovitis (n=11) and healthy joints (n=14) were collected from 24 equine patients and seven slaughterhouse horses. The samples from the joints with non-infectious synovitis and healthy joints served as negative controls. After isolation, counting and identification of neutrophils, the percentage viability, and the proportion apoptotic and necrotic neutrophils were determined by flow cytometry. Viability was significantly higher in infected samples compared to the controls. A significant ...
Brunso L, Segura D, Monreal L, Escolar G, White JG, Diaz-Ricart M.Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cy...
Hackett CH, Flaminio MJ, Fortier LA.A long-term goal of mesenchymal progenitor cell (MPC) research is to identify cell-surface markers to facilitate MPC isolation. One reported MPC feature in humans and other species is lack of CD14 (lipopolysaccharide receptor) expression. The aim of this study was to evaluate CD14 as an MPC sorting marker. Our hypothesis was that cells negatively selected by CD14 expression would enrich MPC colony formation compared with unsorted and CD14-positive fractions. After validation of reagents, bone marrow aspirate was obtained from 12 horses. Fresh and cultured cells were analyzed by flow cytometry ...
Braun J, Hack A, Weis-Klemm M, Conrad S, Treml S, Kohler K, Walliser U, Skutella T, Aicher WK.To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs). Methods: 6 adult racing horses treated for articular Injury but otherwise healthy. Methods: AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli...
Park SB, Seo MS, Kang JG, Chae JS, Kang KS.Amniotic fluid (AF) is a well-known source of stem cells. However, there have been no reports regarding equine AF stem cells. We have isolated equine AF-derived multipotent stem cells (MSC) (eAF-MSC) and show that these cells exhibit self-renewal ability and multilineage differentiation. Methods: AF was obtained from thoroughbred mares and mononuclear cells (MNC) were isolated by Ficoll-Paque density gradient. We measured the cumulative population doubling level (CPDL) and characterized the immunophenotype by flow cytometry. To investigate differentiation ability, a trilineage differentiation ...
Owens SD, Johns JL, Walker NJ, Librach FA, Carrade DD, Tablin F, Borjesson DL.To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. Methods: 13 healthy adult Thoroughbreds. Methods: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfus...
Kabithe E, Hillegas J, Stokol T, Moore J, Wagner B.CD14 is a receptor for the complex of lipopolysaccaride (LPS) and LPS-binding protein. Binding of this complex to CD14 in association with Toll-like receptor 4 provides a major pathway for the initiation of innate immune responses to bacterial pathogens. We used a mammalian expressed extracellular region of equine CD14 (rCD14) derived from an IgG fusion protein to produce monoclonal antibodies (mAbs) to CD14. Eight mAbs were tested by flow cytometric analysis of equine leukocytes and by immunoblotting using rCD14 indicating that the mAbs recognized at least three different epitopes on equine C...
Flores J, Corvera Poiré E, del Rio JA, López de Haro M.We consider a simple model to give a plausible mechanical explanation of what are the actual resting heart rates of mammals optimized for. We study what is the optimal frequency for a viscoelastic fluid circulating in a pulsatile way through a network of tubes and conclude that the heart rate is not optimized to transport blood through the whole net. Rather, actual resting heart rates of mammals happen at frequencies that optimize flow in vessels of radii that correspond to large arteries, which bring oxygenated blood rapidly far away from the heart, towards head and limbs. Our results for the...
Scheinberg P, Marte M, Nunez O, Young NS.Clones of glycosylphosphatidylinositol-anchor protein-deficient cells are characteristic in paroxysmal nocturnal hemoglobinuria and are present in about 40-50% of patients with severe aplastic anemia. Flow cytometry has allowed for sensitive and precise measurement of glycosylphosphatidylinositol-anchor protein-deficient red blood cells and neutrophils in severe aplastic anemia. Methods: We conducted a retrospective analysis of paroxysmal nocturnal hemoglobinuria clones measured by flow cytometry in 207 consecutive severe aplastic anemia patients who received immunosuppressive therapy with a h...
Minervini F, Lacalandra GM, Filannino A, Garbetta A, Nicassio M, Dell'aquila ME, Visconti A.Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm mo...
Dunkel B, Rickards KJ, Werling D, Page CP, Cunningham FM.To determine whether expression of equine platelet activation-dependent surface markers is influenced by phospodiesterase (PDE) isoenzyme activity and whether antigen challenge alters platelet PDE activity in horses with recurrent airway obstruction (RAO). Methods: 16 horses. Methods: 7 healthy horses were used for in vitro experiments, 6 horses with RAO were used for antigen challenge, and 6 healthy horses were used as control animals. Three of the healthy horses had also been used in the in vitro experiments. Effects of PDE inhibition and activation of adenylyl cyclase on CD41/61 and CD62P e...
Wagner B, Burton A, Ainsworth D.Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory TR1 cell phenotypes respectively, by flow ...
Go YY, Zhang J, Timoney PJ, Cook RF, Horohov DW, Balasuriya UB.Extensive cell culture passage of the virulent Bucyrus (VB) strain of equine arteritis virus (EAV) to produce the modified live virus (MLV) vaccine strain has altered its tropism for equine CD3(+) T lymphocytes and CD14(+) monocytes. The VB strain primarily infects CD14(+) monocytes and a small subpopulation of CD3(+) T lymphocytes (predominantly CD4(+) T lymphocytes), as determined by dual-color flow cytometry. In contrast, the MLV vaccine strain has a significantly reduced ability to infect CD14(+) monocytes and has lost its capability to infect CD3(+) T lymphocytes. Using a panel of five re...
Krakowski L, Krawczyk CH, Wrona Z, Dabrowski R, Jarosz Ł.The objective of the present study was to analyse the immune status of mares in the early stages of pregnancy and mares which experienced early embryonic death (EED). Forty-eight fertile purebred Arabian mares were used for the study. The mares were divided into two groups. Group I was the control group composed of 32 mares in the early stages of a normal pregnancy. Group II was composed of 12 mares which had experienced EED. In both groups, subpopulations of T lymphocytes containing CD2(+), CD4(+) and CD8(+) were characterized using flow cytometry. The percentages of CD3(+) lymphocytes (addin...
Platt R, Sponseller BA, Chiang YW, Roth JA.The cell-mediated immune (CMI) response of foals to virulent equine herpesvirus-1 (EHV-1) infection was evaluated by multi-parameter flow cytometry (FCM). Ten 7-8-month-old EHV-1 seronegative foals were infected intranasally with virulent EHV-1 and 10 foals served as uninfected controls. Blood samples were collected 6 and 7 weeks after infection to test for specific CMI responses to live heterologous EHV-1 recall antigen. The activation markers included major histocompatibility complex class II (MHC II), intracellular interferon gamma (IFN-gamma) and interleukin 4 (IL-4). The results from both...
Prieto JMB, Tallmadge RL, Felippe MJB.Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5 B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5 and CD5 B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segment...
Krakowski L, Obara J, Wąchocka A, Piech T, Bartoszek P, Kostro K, Tatara MR.The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. Th...
Durán MC, Willenbrock S, Carlson R, Feige K, Nolte I, Murua Escobar H.CD14 positive (CD14+) cells are the precursor cells of monocyte-derived dendritic cells (DCs). In horses their potent antigen-presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. Objective: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell...
van der Meulen KM, Nauwynck HJ, Pensaert MB.Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-s...
Lalko CC, Deppe E, Ulatowski D, Lutgen A, Hart AP, Patton EA, Lunn DP, Suresh M, Darien BJ.Acute inflammatory diseases, such as colic, septicemia and endotoxemia are common in equines and have been shown to be correlated to vascular injury and thrombosis. In humans with similar thrombotic conditions, P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1)-mediated platelet-leukocyte adhesion contributes to the pathogenesis of these disorders through the generation of inflammatory mediators and tissue factor. As such, we hypothesized that a P-selectin-PSGL-1 (platelet-leukocyte) interaction, similar to that in humans, may also exist in the horse. The objective of this study was to i...
Gardner RB, Nydam DV, Luna JA, Bicalho ML, Matychak MB, Flaminio MJ.Phagocytic activity of neonatal foals has been reported to be similar to that of adult horses, but serum opsonization capacity develops with age and may be further altered when opsonins are consumed during infection. Objective: Phagocytosis, oxidative burst activity, and serum opsonization capacity in neonatal foals admitted to an intensive care unit are reduced in comparison with control foals. Methods: Blood samples were collected from hospitalized neonatal foals and from control foals. Hospitalized foals were characterized as sick or septic on the basis of a sepsis score and received intrav...
Raidal SL, Rose RJ, Love DN.In this study, the effects of prolonged, high intensity training on aspects of peripheral blood and bronchoalveolar lavage (BAL)-derived leucocyte function were evaluated in 8 horses. All horses undertook a 7 week endurance training programme, followed by 5 weeks of high intensity training (HIT). Thereafter, horses were divided into control (C) and overtraining (OT) groups. The frequency and intensity of training were increased more substantially for horses in the OT group. Training was terminated in week 32 when horses in the OT group demonstrated a significant performance reduction. Peripher...
Pickles KJ, Brooks AC, Rickards KJ, Cunningham FM.N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil ...
Lima-Neto JF, Fernandes CB, Alvarenga MA, Golim MA, Landim-Alvarenga FC.This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and pro...
Odeh AI, Dascanio JJ, Caceci T, Bowen J, Eng LA.Phospholipids are an essential component of all mammalian cells; platelet activating factor (PAF=1-O-alkyl-acetyl-sn-glycero-3-phosphocholine) is a signalling phospholipid that has many biological properties in addition to platelet activation. PAF receptors have been detected on stallion spermatozoa; therefore, the aim of this study was to evaluate the effect of synthetic PAF on the motility, capacitation and the acrosome reaction of stallion spermatozoa. Treatment of ten stallion semen samples with 10(-4)-10(-13) mol PAF l(-1) resulted in significant differences in motility and capacitation (...
Balao da Silva C, Macías-García B, Morillo Rodriguez A, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Morrell JM, Rodriguez-Martínez H....The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 μM of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and mem...
Kleiber C, Grünig G, Jungi T, Schmucker N, Gerber H, Davis WC, Straub R.A panel of specific antibodies against CD3, CD4, CD5, CD8, MHC I and II was used in single and two colour flow cytometry to define T cell subpopulations in bronchoalveolar lavage fluid of horses affected with chronic obstructive pulmonary disease and of healthy controls. According to the results of the clinical examination including bronchoscopy and cytology of the tracheal aspirate the horses were divided into four groups (healthy, subclinically to mildly affected; moderately affected, and severely affected). All groups of horses had a similar percentage of CD3+ cells in the BALF. Compared to...
Boere J, van de Lest CHA, de Grauw JC, Plomp SGM, Libregts SFWM, Arkesteijn GJA, Malda J, Wauben MHM, van Weeren PR.Extracellular vesicle (EV) concentration, characteristics and function in equine synovial fluid (SF) during normal growth and development has not previously been studied. Isolation of EVs was performed in SF from three healthy foals and two adult horses by differential ultracentrifugation (10,000g and 200,000g); EVs were purified by sucrose density gradient floatation and analysed by high-resolution flow cytometry (FCM), buoyant density and western blotting. Additionally, repeated biomarker analysis of sulphated glycosaminoglycans (GAG), matrix metalloproteinase (MMP), C-terminal crosslinked t...
Moura TCM, Arruda LCP, Araújo Silva RAJ, Silva RPF, Oliveira AS, Tobal LFM, Batista AM, Carneiro GF, Guerra MMP.The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 10 sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (-196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasm...
Bastos FZ, Barussi FCM, Leite LMB, Jamur VR, Soares AA, Senegaglia AC, Michelotto PV.This study aimed to assess the safety and reproducibility of cell therapy for its use in clinical practice. We performed immunophenotypic characterization of equine bone marrow-derived mesenchymal stromal cells (BMMSCs) by flow cytometry using CD90, CD19, CD14, CD105, CD45, and HLA-DR markers (n = 4); GTG banding cytogenetic analysis (n = 3); and microbiological quality control (n = 4). The immunomodulatory potentials of BMMSCs (n = 4) and its conditioned medium (CM, n = 3) were investigated by in vitro lymphocyte inhibition assay using phytohemagglutinin (PHA)-stimulated peripheral ...
Diaz-Jimenez M, Dorado J, Consuegra C, Ortiz I, Pereira B, Carrasco JJ, Gomez-Arrones V, Domingo A, Hidalgo M.The aim of this study was to assess the effect of different factors affecting vitrification success of donkey sperm: extender, sperm concentration, volume and storage vessel type. In Experiment 1, sucrose supplementations at 0.25 and 0.1 M were compared using two base extenders (containing or not egg-yolk); in Experiment 2, three sperm concentrations were assessed: 100, 200 or 300 million sperm/mL; and in Experiment 3, three different sperm volumes (100, 160 and 200 μL) and two different storage vessels (0.25 and 0.5 mL straws) were assessed. Sperm motility variables (CASA), plasma memb...
Albornoz A, Morales N, Uberti B, Henriquez C, Burgos RA, Alarcon P, Moran G.Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its me...
Sharpe AN, Seeto WJ, Winter RL, Zhong Q, Lipke EA, Wooldridge AA.OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth ...
Hines MT, Palmer GH, Byrne KM, Brassfield AL, McGuire TC.Bronchoalveolar lavage fluid (BALF) and peripheral blood were obtained from each of 17 adult Arabian mares and absolute numbers and relative lymphocyte proportions were determined for total T lymphocytes, using CD2 as a marker, CD4 + T lymphocytes, CD8 + T lymphocytes, CD5 + lymphocytes, and sIgM + B lymphocytes. The marked variation in BALF cell recovery resulted in wide variation in absolute values for each lymphocyte subset. The relative proportions of gated BALF lymphocytes were much less variable and provided a basis for comparison of lymphocyte subsets between the BALF and peripheral blo...
de Vasconcelos Franco JS, Faheem M, Chaveiro A, Moreira da Silva F.The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C...
Tschetter JR, Davis WC, Perryman LE, McGuire TC.Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes w...
Norris JW, Pratt SM, Hunter JF, Gardner IA, Tablin F.To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm. Methods: 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males. Methods: Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen. Results: Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 ...
Springer NL, Smith E, Brooks MB, Stokol T.To develop a flow cytometric assay to quantify platelet-derived microparticles (PMPs) in equine whole blood and plasma. Methods: Citrate-anticoagulated whole blood from 30 healthy adult horses. Methods: Platelet-poor plasma (PPP) was prepared from fresh whole blood by sequential low-speed centrifugation (twice at 2,500 × g). Samples of fresh whole blood and PPP were removed and stored at 4° and 24°C for 24 hours. Platelet-derived microparticles were characterized in fresh and stored samples on the basis of the forward scatter threshold (log forward scatter < 10(1)) and labeling with anne...
Cassart D, Fett T, Sarlet M, Baise E, Coignoul F, Desmecht D.The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical...
Theuerkauf K, Obach-Schröck C, Staszyk C, Moritz A, Roscher KA.In humans, activated platelets contribute to sepsis complications and to multiple organ failure. In our prospective analytical study of cases of the equine systemic inflammatory response syndrome (SIRS), we adapted a standard human protocol for the measurement of activated platelets and platelet-leukocyte aggregates (PLAs) in equine platelet-leukocyte-rich plasma (PLRP) by flow cytometry, and we investigated the hypothesis that activated platelets and PLAs are increased in clinical cases of SIRS. We included 17 adult horses and ponies fulfilling at least 2 SIRS criteria, and 10 healthy equids ...
Ferrer MS, Miller LMJ.Antisperm antibodies (ASAs) have been associated with infertility in stallions. The objectives of this study were to investigate the frequency of ASA-positive semen samples in satisfactory and non-satisfactory breeder stallions, the association between ASA binding and semen quality, and factors that may affect the diagnosis. Breeding soundness examinations were performed in 21 stallions and the percentage of IgG- and IgA-bound spermatozoa was evaluated using flow cytometry. Median IgG and IgA binding did not differ between the first and second ejaculates. The percentage of IgA-bound spermatozo...
Gröndahl G, Johannisson A, Jensen-Waern M.The ability of equine plasma from different donors to enhance phagocytic capacity was assessed in neutrophils obtained from seven foals, aged 7-8 days (Study A), and from seven adult horses (Study B). Neutrophils were allowed to phagocytize fluorescent yeast cells opsonized with plasma from one of three donors or with pooled serum, all previously frozen (-18 degrees C) and thawed. The results were analysed by flow cytometry. In study A, fresh autologous foal serum was also used for opsonization, and in study B, heat-inactivated plasma and pooled serum were used in addition to untreated samples...
Keller LE, Tait Wojno ED, Begum L, Fortier LA.Infiltration of cluster of differentiation (CD) 3 (CD3) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. An imbala...
Mundy LN, Ishihara A, Wellman ML, Bertone AL.To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells. Methods: 12 healthy adult horses. Methods: Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility ...
Siedek EM, Honnah-Symns N, Fincham SC, Mayall S, Hamblin AS.Monoclonal antibodies (mAbs) recognizing equine macrophages are scarce. The present study compared the immunocytochemical staining of various equine tissues (lymphoid tissue, lung, liver, small intestine, skin and blood leucocytes) by an antibody, Ki-M6, which detects CD68 in human macrophages and dendritic cells, and by a new anti-equine mAb, JB10, with staining produced by two previously described anti-equine macrophage mAbs, CZ2.2 and CZ3.3. Ki-M6 was shown to identify equine macrophages, which had a distribution different from those identified by CZ2.2 and CZ3.3. JB10 identified equine mac...
